Marine Biotoxins and Harmful Algae:
A National Plan

II. THE TOXINS

1. General Background

In the United States, the most significant economic and public health problems related
to harmful algae are:

Paralytic shellfish poisoning (PSP), which occurs in all coastal New England states
as well as New York and along much of the west coast from Alaska to California.
This problem has also extended to offshore areas in the northeast (causative species
- the dinoflagellates Alexandrium tamarense
, A. fundyense,
and A. catenella
; Anderson et al., 1982; Nishitani and Chew, 1988; Price and Kizer, 1990).

Amnesic shellfish poisoning (ASP) which occurred first in southeastern Canada in 1987, but has been a problem for the U.S. Pacific coast states over the past two years
(causative species - the diatoms Pseudonitzschia pungens
forma multiseries and Pseudonitzschia australis;
Garrison et al., 1992; Buck et al., 1992; Fritz et al., 1992; Wood and Shapiro, 1992).
This sometimes fatal illness is so named because one of its most severe symptoms
is the permanent loss of short-term memory. The ASP toxin, domoic acid, has been
detected in shellfish from both the West and East Coasts of the United States, and toxic
P. pungens
f. multiseries
cells have been isolated from Gulf of Mexico waters, though no toxin has yet been
detected in the field. Thus, the threat to U.S. shellfish consumers from this dangerous
alga covers a broad geographic area. The name "ASP" understates the severity of
this problem, as it is now known that domoic acid also accumulates in fish and in crab
viscera along the west coast of the United States, where the impact of this toxin
on non-molluscan fisheries may well exceed the loss to molluscan fisheries (e.g.,
razor clam).

Another serious threat is diarrhetic shellfish poisoning (DSP) which some consider
the most serious and globally widespread phytoplankton-related seafood illness.
The first confirmed incidence of DSP in North America occurred in 1990 when these
toxins were detected in shellfish from the southern coast of Nova Scotia following numerous
human illnesses (Quilliam et al., submitted ms.). Another DSP outbreak in Canada
occurred in 1992 (Wright, pers. comm.). DSP-producing species of phytoplankton occur
throughout all temperate coastal waters of the United States, and thus present a potential
problem for the future, though no outbreaks of DSP have yet been confirmed.

2. Toxicology and Pharmacology

2.1 Background

Naturally-occurring toxins responsible for the intoxication syndromes associated with
seafood are as diverse as are the algae that produce them (Table 1). Man is exposed
principally

by consumption of contaminated seafood products, although one type of toxin (brevetoxin),
because of aerosol formation due to wave action, also causes respiratory asthma-like
symptoms during blooms of the toxigenic organism. In all cases, the diseases are caused by specific interaction of the toxins with tissues and organs responsible
for carrying out vital cellular functions. By modifying these functions in deleterious
ways, the toxins disrupt nerve electrical conduction, uncouple communication between
nerve and muscle, and prevent critical physiological processes from occurring. Most
of the toxins accomplish this by binding to specific receptors, or docking sites,
on the tissue or organ leading to critical changes in intracellular concentration
of ions such as sodium, calcium, and potassium. Some of the cellular changes lead to permanent
effects in the exposed cells.

Seafood toxins bind with high affinity to specific receptor sites, often with binding
constants in the 10-9 to 10-12 M range. Most binding is reversible, but dissociation times may be quite prolonged.
Except for identification of the general category of toxin receptors in living organisms,
virtually nothing is known about the chemical interaction of the toxins with their specific binding sites.

Many of the toxin classes are not single chemical entities, but instead represent
families of compounds of similar chemical structure (Table 1). Each toxin derivative
of the same parent compound is slightly altered in chemistry. This leads to wide-ranging variability in toxicity of the individual modified toxins.

Acute single-dose lethality of seafood toxins has been extensively studied in the
laboratory (Shimizu, 1987). However, chronic and/or repeated exposure to marine
seafood toxins, which is a more realistic phenomenon, has not been adequately examined.
There is a serious lack of knowledge as to how the toxins are distributed throughout the
body and eliminated. Other important questions include how long the toxins circulate
before elimination, and how they are metabolized by living organisms. These knowledge gaps prevent researchers from devising antidotes or effective treatments which may
alleviate or lessen the symptoms. Therapeutic intervention is primarily limited
to symptomatic treatment and life support if necessary.

Similarly, statistical data collection on human exposure, intoxication duration, and
number of incidences are limited and incomplete. Many cases of intoxication are
not reported, or are reported inadequately based on hear-say evidence with little
documentation.

Since 1978, illnesses in the U.S. due to natural algal toxins have included CFP, PSP,
NSP and ASP. No incidents of DSP have yet been verified in this country. Although
records are incomplete because reporting to the Centers for Disease Control (CDC)
is voluntary, evidence indicates that ciguatera was responsible for about half of all
seafood intoxications between 1978 and 1987 (Ahmed, 1991). A growing body of evidence
indicates that incidents of ASP are on the increase (Buck et al., 1992; Garrison
et al., 1992; Villac et al., in press; Horner and Postel, in press), and that DSP may shortly
make its début in the United States. Certain of the toxicoses, like the toxigenic
organisms, are focused geographically and result from consumption of particular species. However, with the increase in interstate and international transport of seafood,
as well as international travel by seafood consumers, there are virtually no human
populations that are free from risk.

ASP:
Amnesic shellfish poisoning
can be a life-threatening syndrome. It is characterized by both gastrointestinal
and neurological disorders (Bates et al., 1989). Gastroenteritis usually develops
within 24 hours of the consumption of toxic shellfish; symptoms include nausea, vomiting,
abdominal cramps, and diarrhea. In severe cases, neurological symptoms also appear,
usually within 48 hours of toxic shellfish consumption. These symptoms include dizziness,
headache, seizures, disorientation, short-term memory loss, respiratory difficulty, and coma. In 1987, four victims died after consuming toxic mussels from Prince
Edward Island, Canada. Since that time, Canadian authorities have monitored both
the water column for the presence of the causative diatom, and shellfish for the
presence of the toxin, domoic acid. Shellfish beds are closed to harvesting when the domoic
acid concentration reaches 20 g/g shellfish meat. Fish and crab viscera can also
contain domoic acid, so the risk to human consumers and animals in the marine food
chain is more significant than previously believed.

Ciguatera:Ciguatera fish poisoning
produces gastrointestinal, neurological, and cardiovascular symptoms. Generally,
diarrhea, vomiting, and abdominal pain occur initially, followed by neurological
dysfunction including reversal of temperature sensation, muscular aches, dizziness,
anxiety, sweating, and a numbness and tingling of the mouth and digits. Paralysis and death
have been documented, but symptoms are usually less severe although debilitating
(Miller, 1991). Recovery time is variable, and may take weeks, months, or years.
Rapid treatment (within 24 hours) with manitol is reported to relieve some symptoms. There
is no antidote, supportive therapy is the rule, and survivors recover. Absolute prevention
of intoxication depends upon complete abstinence from eating any tropical reef fish, since there is currently no easy way to measure routinely ciguatoxin or maitotoxin
in any seafood product prior to consumption.

DSP: Diarrhetic shellfish poisoning
produces gastrointestinal symptoms, usually beginning within 30 min to a few hours
after consumption of toxic shellfish (Yasumoto and Murato, 1990). The illness, which
is is not fatal, is characterized by incapacitating diarrhea, nausea, vomiting, abdominal cramps, and chills. Recovery occurs within three days, with or without medical
treatment.

NSP: Neurotoxic shellfish poisoning
produces an intoxication syndrome nearly identical to that of ciguatera. In this
case, gastrointestinal and neurological symptoms predominate. As noted above, formation
of toxic aerosols by wave action can produce respiratory asthma-like symptoms. No
deaths have been reported and the syndrome is less severe than ciguatera, but nevertheless
debilitating. Unlike ciguatera, recovery is generally complete in a few days. Monitoring
programs (based on G
. breve
cell counts) generally suffice for preventing human intoxication, except when officials
are caught off-guard in previously unaffected areas.

PSP: Paralytic shellfish poisoning
, like ASP, is a life threatening syndrome. Symptoms are purely neurological and
their onset is rapid. Duration of effects is a few days in non-lethal cases. Symptoms
include tingling, numbness, and burning of the perioral region, ataxia, giddiness,
drowsiness, fever, rash, and staggering. The most severe cases result in respiratory
arrest within 24 hours of consumption of the toxic shellfish. There is no antidote,
supportive therapy is the rule and survivors recover. PSP is prevented by large-scale proactive monitoring programs (assessing toxin levels in mussels, oysters, scallops,
clams) and rapid closures to harvest of suspect or demonstrated toxic areas.

2.2. Impediments and Recommendations

The Toxins
working group identified three major impediments to progress in the area of toxin
pharmacology and toxicology, and recommended solutions to these impediments. Priority
order was not assigned to the impediments.

IMPEDIMENT: Reference toxin is difficult to obtain, is not always reproducible, and
is generally costly.
This impedes development of methods for detection, prevents detailed studies of physiology,
and inhibits development of molecular pharmacology to explain toxin interaction at
the receptor level.RECOMMENDATION: Establish reference toxin supplies for the five major classes of
toxins.
This can be accomplished by isolation and purification of toxin from fish and shellfish,
from mass cultures of the toxic phytoplankton, or synthesis of less accessible toxins.
Production of radiolabeled toxin first requires adequate supplies of purified standards. Three levels of standards are required: Pharmacological Standards, Analytical
Standards, and Certified Standards.

Toxic marine dinoflagellates are some of the most difficult algae to grow in mass
culture. Facilities required are extensive and sophisticated, and careful control
of nutrient levels, pH, temperature, lighting, security, and cleanliness is necessary.
Likewise, the facilities necessary to extract and purify multi-milligram quantities of
these highly potent materials are extensive, and require special instrumentation
for separation, isolation, detection and storage. At many such facilities, detailed
safety plans are implemented. Although this is an objective which should be achieved as rapidly
as possible, it must be recognized that a continuing financial commitment is necessary
to implement the production of consistent, reliable standards. With the exception of ciguatera-related toxins, facilities expansion to produce the desired quantities
of all levels of standards is a logistical possibility within 2-3 years. The ciguatera
toxins will require extensive research prior to standards availability (see toxin
standards section).

A major problem with available biotoxin supplies is their distribution to monitoring
agencies and the research community. A well-defined distribution plan for biotoxin
standards isolated or synthesized with the assistance of federal funds needs to be
prepared. It is an overwhelming consensus that toxins should be made available at a minimal
cost, for example, as is done by the National Hormone and Pituitary Program.

Without knowledge of the actual magnitude of the problem, little can be done to evaluate
remedial measures aimed at reducing incidence. Effects of episodic and chronic exposure
have been totally neglected. It is also apparent that part of the reporting problem is due to lack of toxic syndrome recognition.

RECOMMENDATION
: Develop a database in collaboration with the CDC for marine seafood intoxication.
Explore development of better reporting tools, including mandatory reporting. Incorporate
episodic or chronic exposures. Educate physicians, public health officials, and consumers in issues of seafood poisoning.

Incidences of seafood intoxication are thought to far exceed the actual reporting.
Valuable information regarding seafood intoxication does not reach the proper reporting
officials for many reasons. Initial symptoms often are gastrointestinal in nature
and are misdiagnosed as the "flu." Only later, after intensification of symptoms or
a significant number of individual events, are alternative diagnoses considered.
Early identification of the toxicologic syndrome is necessary to permit effective
therapeutic intervention. Once recognized, proper and prompt reporting alerts official agencies
to implement regulatory directives. If the syndromes are recognized, and the reporting
is mandatory, the data will be comprehensively recovered. This is a long-term objective, and has appended to it a continuing effort to collect and analyze data, to
educate, and to develop the program further. Initial funds (1-2 yrs) should be provided to trained
epidemiologists and biostatisticians for examining the status of the situation and
providing additional recommendations for full implementation of a program. Ideally,
this should be undertaken by an epidemiologist or group of epidemiologists with interest
and experience in marine toxin poisoning. Full implementation of this program would
take 5-10 years.

RECOMMENDATION: Identify primary tissues of toxicologic action in animals and man,
and develop in vitro
models that reflect the primary toxicologic action. Identify the molecular characteristics
of specific receptors for saxitoxin/tetrodotoxin, brevetoxin/ciguatoxin, maitotoxin,
okadaic acid, and domoic acid. Explore structural modification of toxins in relation to toxicity and binding affinity.

The molecular mechanism of intoxication is not known in detail for any marine toxin.
This is in large part due to the fact that we do not know the primary targets for
the biotoxin in the human (or animal) body. It is critical to define the primary
relevant site of action for marine biotoxins, before funding substantial amounts of research
on nonrelevant tissues.

Laboratory studies which employ radiolabeled toxins are capable of defining specific
binding or recognition sites on a molecular level. Detailed knowledge of the toxin
receptors is essential for understanding why "toxins" are toxic. Elucidation of the
mechanism of action provides information essential for the development of receptor-based
methods for detection, and practical methods for treatment of intoxication. Receptor-derived
assays correlate well with toxicity, and may be enhanced using molecular recombinant technology, thereby reducing the need for animal-based toxicological assays.
With our current capabilities and available molecular probes, brevetoxin research
will continue (complete in 2-3 years), and pharmacologic probe synthesis and investigation initiated for saxitoxin, okadaic acid, and domoic acid (3-6 yrs). Maitotoxin
and ciguatoxin(s) will require more structural information prior to direct analysis
(> 7 yrs).

3. Analysis, Standards, Chemistry

3.1. Background

The chemical structures of ASP, DSP (okadaic acid family), NSP and PSP toxins are
known (Baden, 1984; Shimizu, 1984; Yasumoto et al., 1984; Shimizu et al., 1986; Wright
et al., 1989; Hall and Strichartz, 1990). Structures of the toxins responsible for
ciguatera are not yet known, except for ciguatoxin in fish from the Tahitian region (Murata
et al., 1990). In addition, the structure and role of maitotoxin (MTX) is only partially
understood (Murata et al., 1992), as is its role in ciguatera fish poisoning. Determination of the structures of newly-recognized marine biotoxins requires the
dedication of substantial fiscal resources, instrumentation, and personnel time.
Currently, the only certified standard(s) available is for domoic acid (Institute
for Marine Biosciences, National Research Council of Canada, Halifax, NS, Canada). Reference
material is also available for domoic acid. Moreover, suites of standards for the
different naturally-occurring toxins are necessary.

Biological tests using mice or rats are available for all listed toxins, but the bioassay
for ASP toxins lacks sufficient sensitivity (Quilliam et al., 1989). Other biological
tests that do not use animals have yet to be subjected to collaborative review. Accepted chemical analytical methods have been developed only for the ASP toxin,
domoic acid (Quilliam, et al., 1991; Pocklington, et al., 1990). Chemical methods
exist for the other toxins but have not gone through appropriate collaborative review
(e.g., for PSP) or need further refinement (CTX, DSP, NSP).

In recent months, the detection of known toxins in unexpected vectors (e.g., PSP toxins
in crab viscera, ASP toxins in fish and crab viscera) has accentuated deficiencies
in assay procedures. The extension of certain assay procedures to "new" tissues can
sometimes be done routinely when the organisms are related, but new procedures need
to be developed when organisms not covered by the original method are investigated.
Substitution of marine mammal liver for clam tissue in PSP testing, for example,
can lead to erroneous conclusions.

3.2. Impediments and Recommendations

The Toxins
working group identified three major impediments to progress associated with the
development of standards and assays and provided recommended solutions to these non-prioritized
impediments.

IMPEDIMENT: The chemical complexity of marine biotoxins impedes the development of
a viable seafood safety program and successful fisheries resource management. RECOMMENDATION: Determine the structure, chemical properties, and pharmacological
behavior of marine toxins. Develop methods for extraction and purification of toxins
from natural sources, their synthesis, and preparation of toxin derivatives.

The determination of the structure, properties, and behavior of marine toxins is an
essential first step for the development of new and improved methods for the detection
of toxins, for mitigating their presence and effects in seafoods, and for a successful fisheries resource management program. This expanded knowledge will augment the
characterization of new seafood toxins as they arise. The structural characterization
of marine toxins has challenged researchers for many years due to lack of appropriate
expertise, resources, and specialized equipment and techniques not commonly available,
such as nuclear magnetic resonance (NMR) spectrometers and "soft" ionization mass
spectrometers.

Mass culture of toxic algae, chemical synthesis, chemical modification, and derivatization
methods are necessary for the economical production of toxin standards and related
compounds, and are vital for the preparation of easily-detected derivatives for use in monitoring programs and toxicological studies. Toxicological and pharmacological
studies are necessary for the development of antidotes or other medical intervention
techniques. These activities might require 3-5 years for known toxins, and 5-7 years for unknown or poorly understood toxins.

IMPEDIMENT: Every study related to marine biotoxins is compromised by a lack of toxin
standards. It is impossible to develop methods for the detection and quantification
of marine toxins without the availability of defined marine toxin standards.RECOMMENDATION: Isolate or synthesize, and characterize sufficient quantities of
purified toxin(s) for the development of appropriate standards. Establish a toxin
standard development, maintenance, and distribution system, so that these toxins
are equally accessible and available to all qualified research groups
.

Biological, toxicological, and chemical studies require submilligram to milligram
quantities of rigorously characterized marine toxins. This work will require the
collaboration of biologists to produce adequate quantities of source materials, and
chemists to isolate, purify, and characterize the toxins. Once prepared, either by biological
or synthetic means, these toxin standards must be readily and reliably available
to research and monitoring programs so that public health is adequately protected
and fisheries resources are effectively managed. These standards should have international
acceptance.IMPEDIMENT: Current assay methods for marine biotoxins are inadequate for most monitoring
and research purposes. Most monitoring programs utilize whole animal assays which
lack sensitivity and are becoming increasingly unacceptable.

RECOMMENDATION: Develop rapid and cost-effective methods for detecting and quantifying
marine toxins that are internationally
acceptable.

The seafood industry is a global activity that requires coordination of safety regulations
between trading nations. To avoid conflicts, the development of marine toxin action
levels and methods must be evolved in close cooperation with other international agencies. A particularly important challenge is the development of rapid field methods,
such as enzyme or immunoassay-based kits. Sensitivity of all methods must relate
to human toxicity and to levels present in contaminated seafoods. The methods developed must be subjected to verification through formal, collaborative studies.